Maxwell rsc simplyrna tissue kit

Manufactured by Promega

Sourced in United States, Germany, Japan

The Maxwell RSC simplyRNA Tissue Kit is a laboratory equipment product designed for the automated extraction and purification of RNA from tissue samples. It provides a streamlined and efficient solution for isolating high-quality RNA from a variety of tissue types.

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Maxwell simplyRNA Tissue kit (9 citations) RSC simplyRNA Tissue (2 citations) Maxwell SimplyRNA kit (12 citations) SimplyRNA Tissue Kit (11 citations) Maxwell RSC (60 citations) Maxwell kits (2 citations)

107 protocols using maxwell rsc simplyrna tissue kit

Viral Genome Analysis of RNA-seq Data

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RNA was extracted from snap-frozen tissue of four patients (Maxwell^® RSC simplyRNA Tissue Kit(AS1340), Promega, Southampton, UK). RNA concentration and quality were analyzed using the RNA Nano Kit for the 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA). RNA-sequencing was undertaken by Edinburgh Genomics (University of Edinburgh, Edinburgh, UK). An automated TruSeq stranded mRNA-seq library preparation from total RNA and the NovaSeq sequencing-system was used (100 bp paired-end; 1,750M+1,750M reads, Illumina, San Diego, CA, USA). This RNA sequencing dataset was generated in an independent collaboration with Transgene, and the fact that Transgene funded the RNA sequencing for four cases of the study, did not influence the study design, execution, and results interpretation.
The “viral genome level analysis module” of the bioinformatics pipeline viGen (31 (link)) was used to align the FASTQ files against the human reference genome (hg19), filter out human RNA-sequences and map un-aligned reads against the viral reference. Access to the raw and processed data of this RNA sequencing set is possible via the gene expression omnibus (GEO accession number: GSE160008).

von Witzleben A., Currall E., Wood O., Chudley L., Akinyegun O., Thomas J., Bendjama K., Thomas G.J., Friedmann P.S., King E.V., Laban S, & Ottensmeier C.H. (2021). Correlation of HPV16 Gene Status and Gene Expression With Antibody Seropositivity and TIL Status in OPSCC. Frontiers in Oncology, 10, 591063.

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Quantification of Nutrient Transporter Genes

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RNA was extracted using the Maxwell RSC simply RNA Tissue kit (Promega). cDNA was synthesized from 2 μg of RNA using Revertaid reverse transcriptase with oligo-dT and random hexamers according to manufacturer's instructions (Thermo Scientific). The qPCR was performed using FAST-cycling parameters on a Viia7 (Life Technologies) using SYBRgreen CFX reagent (Life Technologies). Forward and reverse primers for qPCR were obtained from Eurogentec with the following sequences: SLC38A2, 5′-TCCTGTTAAGTGGTGTACTGGT-3′ 5′-CCAGGTG CATTGTGTACCCA-3′; SLC38A5, 5′-CAGGCATCCGA GCCTATGAG-3′ 5′-CCCCAACATTGTGCAGACAG-3′; SLC1A5, 5′-GAGCTGCTTATCCGCTTCTTC-3′, 5′-GGG GCGTACCACATGATCC-3′; SLC6A19, 5′-ACAGGGT ATGTGGACGAGTG-3′; 5′-GTGGAGATGTTGAGCG TCTCT-3′; SLC6A14, 5′-ACCGTGGTAACTGGT CCAAAA-3′, 5′-CGCCTCCACCATTGCTGTAG-3′; SLC6A15, 5′-GGAACTCTCTGTGGGTCAAAG-3′, 5′-CCGCCCAGTTTAGGGCTTA-3′; SLC7A8, 5′-AGGC TGGAACTTTCTGAATTACG-3′, 5′-ACATAAGCGAC ATTGGCAAAGA-3′; SLC38A1, 5′-AACCTCCTTAG GCATGTCTGT-3′, 5′-GCAAAGGCGAGTCCCAA AAT-3′; SLC38A3, 5′-GGAGCTGTATGGAGGG CAAG-3′, 5′-GAACACTGACATCCCGAATGAT-3′; SLC7A6, 5′-ATCGGGATTGCCCTTTCTGG-3′, 5′-CTG GTGATAGCAGCCAGGAC-3′′; SLC38A7, 5′-AAGAG CAGGTTGAGAAGAGTCC-3′; 5′-GAGCCTTCTTCC TGCAAGGT-3′.

Polet F., Martherus R., Corbet C., Pinto A, & Feron O. (2016). Inhibition of glucose metabolism prevents glycosylation of the glutamine transporter ASCT2 and promotes compensatory LAT1 upregulation in leukemia cells. Oncotarget, 7(29), 46371-46383.

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Skin RNA Extraction and qRT-PCR Analysis

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The total RNA was extracted from skin tissue samples using the Maxwell^® RSC SimplyRNA Tissue Kit (Promega) according to the manufacturer’s protocol. Quantitative analysis was performed using real-time RT-PCR in accordance with the MIQE Guidelines using the PowerSYBR Green PCR Master Mix on a QuantStudio 5 (Applied Biosystems by Thermo Fisher Scientific, Rockford, IL, USA). Primer Express software, v3.0.1 (PE Applied Biosystems, Foster City, CA, USA) was used to design the PCR primers. Two widely used reference genes, namely ß-2-microglobulin (ß2-MG) and RPL-38, were tested. The latter gene exhibited the most stable expression levels, and this gene was used as the housekeeping gene. Primers were produced by the custom oligonucleotide synthesis service TIB Molbiol (Berlin, Germany). The well-established comparative −ΔΔCT method was used. Target mRNA expression was normalized between test samples based on the corresponding RPL38 mRNA transcript levels in each skin sample.

Gambichler T., Harnischfeger F., Skrygan M., Majchrzak-Stiller B., Buchholz M., Müller T, & Braumann C. (2023). In Vitro Experiments on the Effects of GP-2250 on BRAF-Mutated Melanoma Cell Lines and Benign Melanocytes. International Journal of Molecular Sciences, 24(20), 15336.

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Quantifying Viral Shedding in Avian Influenza

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To assess viral shedding, the OP and CL swabs collected at 1, 3, 5, 7, 10, and 14 dpi were suspended in 1 mL PBS. Viral titers in the tissues from H9N2 AIV-infected birds were measured using the centrifuged tissue homogenates (10%, w/v). Briefly, 200 μL of the suspension (supernatant) was subjected to RNA extraction using the Maxwell RSC simplyRNA Tissue kit and a Maxwell RSC 48 instrument (Promega, Germany). The cycle threshold (Ct) value was used to calculate the viral load after real-time reverse transcriptase-PCR (rRT-PCR) of the M-gene (Spackman et al., 2003 (link)).
To convert the Ct values to infectious units, quantitative viral standards (ranging from 10^6.0 to 10^0.0 EID₅₀/0.1 mL) of each virus were prepared in egg allantoic fluid. Viral RNA was extracted from these standards and quantified by rRT-PCR. The resulting standard curves showed a high correlation (r² > 0.99) and were used to convert Ct values to EID₅₀ equivalents/0.1 mL. The detection limit for each virus was 10^1.0 EID₅₀/0.1 mL, with Ct values of 39 for LBM261/20 and 38 for LBM284/18 (Supplementary Figure 1). If the Ct value was >35, swabs were retested by isolating virus as described below.

Kye S.J., Park M.J., Kim N.Y., Lee Y.N., Heo G.B., Baek Y.K., Shin J.I., Lee M.H, & Lee Y.J. (2021). Pathogenicity of H9N2 low pathogenic avian influenza viruses of different lineages isolated from live bird markets tested in three animal models: SPF chickens, Korean native chickens, and ducks. Poultry Science, 100(9), 101318.

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RNA Isolation and cDNA Synthesis from Drosophila Tissues

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Male and female flies were separated and flash frozen in liquid nitrogen and then briefly vortexed to separate heads from bodies. After re-frozen in liquid nitrogen, heads and bodies were collected separately in 1.5mL Eppendorf tubes. Total RNA was isolated following the Maxwell® RSC simplyRNA Tissue Kit (Cat.# AS1340) protocol (Promega). cDNA was generated from isolated total RNA as described in the TaKaRa PrimeScript™ 1^st Strand cDNA Synthesis Kit (Clontech) protocol, reagents below were included in the cDNA synthesis kit. Briefly, concentration of isolated total RNA was measured and same amount of total RNA was added to a single PCR tube each. Nuclease free water was added to each tube to achieve the total sample volume of 8 ul and then 1ul of Random 6 mers (50 μM) and 1 ul of dNTP (10 mM) Mixture were added to the RNA sample. The tubes were placed in an Eppendorf thermal cycler and heated at 65° C for 5 minutes and then cool at 4° C until next step. Reaction mixture was prepared with 4.5 ul of RNase free H₂O, 4 ul of 5X PrimeScript Buffer, 0.5 ul of RNase Inhibitor (40 U/μL), and 1 ul of PrimeScript RTase (200 U/μL) and then added to the sample. The sample was placed in the thermal cycler and incubated at 30° C for 10 minutes and at 42° C for 50 minutes and then heated at 70° C for 15 minutes. PCR was then performed to amplify each Pasilla isoform in the cDNA pool.

Gill J., Park Y., McGinnis J., Perez-Sanchez C., Blanchette M, & Si K. (2017). Regulated intron removal integrates motivational state and experience. Cell, 169(5), 836-848.e15.

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SLC10A7 Gene Expression Analysis

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Cells were harvested, and total mRNA was extracted by Maxwell RSC simplyRNA Tissue Kit (Promega). Complementary cDNA was synthesized from 1 µg total RNA by using 8 µl of RT-mix SuperScript III Reverse Transcriptase (Invitrogen). For quantitative expression analysis of the SLC10A7 transcripts, the gene-specific TaqMan Gene Expression Assay Hs04397477_m1 (Thermo Fisher Scientific) was used. The assay Hs02758991_g1 (ThermoFisher) was used for control amplification of GAPDH. The plates were heated for 2 min at 50 °C, 10 min at 95 °C, and then 40 cycles of 15 s at 94 °C and 60 s at 60 °C were applied. All data were expressed as fold changes using the 2^−ΔΔCt method.

Karakus E., Wannowius M., Müller S.F., Leiting S., Leidolf R., Noppes S., Oswald S., Diener M, & Geyer J. (2020). The orphan solute carrier SLC10A7 is a novel negative regulator of intracellular calcium signaling. Scientific Reports, 10, 7248.

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Transcriptome Analysis of Spinal Cord at Cytokine Injection Site

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Transcriptional analysis was performed at 14 DPI. Spinal cords were sampled with a width of 4.5 mm (centered on cytokine injection site) after transcardial perfusion with ice-cold PBS, then immediately frozen with liquid nitrogen and stored at −80°C until extraction. Total RNA extraction was performed using the Maxwell^® RSC simplyRNA Tissue Kit (Promega) according to the manufacturer’s protocol. Microarray analysis was performed using Mouse Genome 430 2.0 Array (Thermo Fisher Scientific) according to the manufacturer’s protocol. Single array analysis was performed using Microarray Suite version 5.0 (MAS5.0) and global scaling as the normalization method. Differential expression analysis and gene set enrichment analysis (GSEA) were performed using R-packages limma (28 (link)) and clusterProfiler (29 (link)) respectively. The gene set libraries for GSEA (Supplementary File 1) were prepared from Enrichr (30 (link)), CellMarker (31 (link)), and Munji et al. reported genes (32 (link)).

Hirata T., Itokazu T., Sasaki A., Sugihara F, & Yamashita T. (2022). Humanized Anti-RGMa Antibody Treatment Promotes Repair of Blood-Spinal Cord Barrier Under Autoimmune Encephalomyelitis in Mice. Frontiers in Immunology, 13, 870126.

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RNA Sequencing and RNA Editing Analysis

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RNA extraction was performed with Maxwell® RSC simplyRNA Tissue Kit (Promega) and Proteinase K. Libraries were prepared by GENEWIZ from total RNA using poly(A) enrichment of the mRNA to remove ribosomal RNA. MGI tech DNBSEQ-G400 (2 × 150 bp), and Illumina Novaseq 6000 sequencing was performed by GENEWIZ for two and one replicate, respectively. Reads were mapped to the GRCh38.105 reference genome with STAR^{51 (link)} (v2.9.7, parameters—quantMode TranscriptomeSAM—outFilterType BySJout—outFilterMultimapNmax 1—outSAMstrandField intronMotif). RNA editing candidate sites were analyzed with REDItools^{52 (link)} (v1.2.1 parameters: -t 13 -e -d -l -U [AG,TC,CT,GA] -p -u -m 20 -T 6-0 -W -v 10 -n 0.05 -g 2 -s 1). Any significant sites identified in the transfected cells with the empty vector were considered as artifacts or SNPs. The editing sites were considered significant if the read count was above 10, the editing frequency different than 1 and the p-value calculated by Fisher’s Exact Test was below 0.05 after correction for multiple tests using the Benjamini–Hochberg correction. The editing sites corresponding to known SNP positions were filtered with the NCBI SNP database (v151).

Ichinose M., Kawabata M., Akaiwa Y., Shimajiri Y., Nakamura I., Tamai T., Nakamura T., Yagi Y, & Gutmann B. (2022). U-to-C RNA editing by synthetic PPR-DYW proteins in bacteria and human culture cells. Communications Biology, 5, 968.

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RNA Isolation and qRT-PCR Analysis

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Total RNA was isolated from transplanted tumors or endothelial cells from the mouse brain using the Maxwell RSC simplyRNA Tissue Kit (Promega Corp.). cDNA was synthesized using the SuperScript III First-Strand Synthesis Kit (Life Technologies). Quantitative real-time RT-PCR was performed using the StepOnePlus system (Applied Biosystems). The primers used for real-time RT-PCR are listed in Supplementary Table 1. The comparative Ct method was used to analyze the relative gene expression. Two independent experiments were performed in duplicate in each experiment. Beta-actin was used as a housekeeping gene for normalization.

Kinoshita T., Tomita H., Okada H., Niwa A., Hyodo F., Kanayama T., Matsuo M., Imaizumi Y., Kuroda T., Hatano Y., Miyai M., Egashira Y., Enomoto Y., Nakayama N., Sugie S., Matsumoto K., Yamaguchi Y., Matsuo M., Hara H., Iwama T, & Hara A. (2021). Endothelial cell-specific reduction of heparan sulfate suppresses glioma growth in mice. Discover. Oncology, 12, 50.

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Northern Blot Analysis of RNA

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Northern blot was performed, as described in ref. [40 ]. Briefly, the total RNA was isolated using the Maxwell RSC simplyRNA tissue kit (Promega, Madison, WI, USA). Then, 10 µg of RNA was run in 1.2% agarose formaldehyde gels. After the run, the gel was treated with 50 mM NaOH for 20 min and transferred onto a Hybond NX neutral nylon membrane (GE Healthcare, Chicago, IL, USA). Hybridization to radiolabeled 5′-(TAACCC)₆-3′ oligonucleotide was performed overnight at 37 °C. The membranes were washed, exposed to a phosphorimager screen, and subsequently imaged on a Typhoon Imager (GE Healthcare, Chicago, IL, USA).

Rosso I, & d’Adda di Fagagna F. (2020). Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR. International Journal of Molecular Sciences, 21(24), 9774.

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